This application is based on application No. 2000-52914 filed in the Korean Industrial Property Office on Sep. 7, 2000, the content of which is incorporated hereinto by reference.
(a) Field of the Invention
The present invention relates to a method for removing endotoxin from the samples containing useful proteins.
(b) Description of the Related Art
One of the methods of mass production for basic proteins having medical activity is that the proteins are produced by using the recombinant microbial expression system. When using the recombinant microbial expression system, especially the system by using the bacteria as a host such as gram (xe2x88x92) bacteria, it is essential to sufficiently remove endotoxin which is a component of cell wall of the host in the purification step.
Endotoxin is a lipopolysaccharide in the cell wall of most gram-negative bacteria such as E.coli. Endotoxin included in proteins is known to cause symptoms of high fever, endotoxin shock, and inflammation even in a very small amount.
1) ion exchange chromatography, 2) affinity chromatography, 3) ultrafiltration, and 4) phase separation using surfactant have been presented to remove the endotoxin.
However, positively-charged basic proteins such as DNA binding proteins can bind to negatively-charged endotoxin through the electrostatic attractive force. Accordingly, Endotoxin cannot be sufficiently removed by ion exchange chromatography or affinity chromatography (Journal of Immunological Methods, vol. 132, pp.191-195, 1990).
Generally, in the ultrafiltration method, endotoxin is removed by using the membrane with 10K cut-off where molecules with smaller molecular weight than 10K pass through the membrane but endotoxin having greater molecular weight remain. However, in case that the material of interest to be removed is macromolecules such as protein, the ultrafiltration method is disadvantageous in that proteins can be lost and the removal efficiency is decreased, because the ultrafilteration process is repetitively preformed by using the membrane with 100K cut-off or more.
The removal method of endotoxin using the phase separation with surfactant is performed by adding surfactant to the protein solution, mixing the resultant, inducing the separation by centrfuging, and then removing the layer containing endotoxin. Though the above method has advantages in high removal efficiency of endotoxin, the disadvantageous need of additional steps is required to remove residual surfactant and the removal process should be performed repetitively. Thus, the yield of purification is decreased. In addition, considering that the temperature of solution must be maintained at 35xc2x0 C. or higher in the method, it is not applicable to temperature-sensitive proteins.
U.S. Pat. No. 5,747,663 disclosed the method of removing endotoxin from the substrate solution where cell lysate was treated with triton X-114 and then with anion exchange chromatography. The endotoxin of 10-12 I.U/mg is still in the resultant solution.
The present invention provides a method whereby endotoxin is effectively removed to an extremely small amount, when the basic protein is separated or purified from the recombinant microorganism.
The present invention relates to the method of removing endotoxin from the sample solution containing the same. The method of present invention comprises the steps of:
(a) adding a detergent to solution containing the basic protein obtained from the recombinant microorganism, and mixing the resultant;
(b) loading the resulting solution on cation exchange resin;
(c) washing the cation exchange resin with solution which does not contain the detergent; and
(d) eluting the basic protein of interest from the cation exchange resin.
By using the method of the present invention, endotoxin can be sufficiently removed from the basic proteins designated therapeutic use.
The method of the present invention is applicable to basic proteins or basic peptides which are purified partially or completely. Thus, depending on the need, the method can be used in the middle step or the final step of purification process.
The term, basic proteins, described herein is intended to mean a peptide or a protein which has the isoelectic point of 7.0 or higher. For examples, the basic proteins include the DNA-binding proteins such as histone, basic peptides with antibacterial activity such as buforin and magainin, and basic fibroblast growth factors, and the like.
The detergent for the present invention is preferably non-ionic detergent including Triton X-114, Triton X-110, Tween 80, Tween 20, and the like. The concentration of the detergent can be 0.01xcx9c10%(v/v), or more preferably 0.1xcx9c2.0%(v/v). When the concentration of detergent is lower, the removal efficiency of endotoxin is decreased. When the concentration is higher, it takes much time to remove residual detergent.
The cation exchange resin applicable to the present invention has no special limitation, and can be selected suitably depending on the kinds of basic proteins to be separated or purified. For examples, the cation exchange resin includes Streamline SP, CM-Sepharose FF, SP-Sepharose FF, Porus 20 HR, and the like.
As needed, to facilitate the separation of endotoxin from the basic protein solution, the solution containing basic protein is mixed with the added detergent, and then, the resulting solution can be left for a time period. The standing time and temperature can be determined depending on the kinds and amount of detergents, and the properties of the basic proteins of interest. In addition, the standing temperature can be determined so that the basic protein solution is not frozen and the activity of protein is maintained. For examples, the temperature is 0xcx9c40xc2x0 C. for histone.
After protein solution is loaded on cation exchange column, the cation exchange column is washed with the buffer solution which does not contain detergent. Thus, it is possible to remove endotoxin and detergent which are contained in the protein solution from the cation exchange column. In such step, the buffer solution for washing the cation exchange column can be the same as or different from that containing the basic proteins.
The buffer solution for eluting basic proteins of interest from the cation exchange column is different from that washing the column.
The medium composition of the present invention is as follows:
RP medium (pH 6.8): KH2PO4 3 g/L, KHPO4 3 g/L, (NH4)2SO4 4 g/L, trisodium citrate 2.3 g/L, MgSO4 0.22 g/L, trace metal solution 10 m/L, Kanamycin 0.1 g/L.
Trace metal solution: ferrite citrate (III) 7.3 g/L, cobalt chloride 0.534 g/L, manganese chloride 3.2 g/L, copper chloride 0.302 g/L, boric acid 0.666 g/L, sodium molybdate 0.534 g/L, zinc acetate 1.687 g/L, thiamine 0.534 g/L, calcium chloride 11.5 g/L, trisodium citrate 20 g/L.
RS medium: RP medium, casamino acid 2 g/L
The present invention is further explained in more detail with reference to the following examples. These examples, however, should not in any sense be interpreted as limiting the scope of the present invention.